High mobility group box proteins (HMGBs) or high mobility group proteins (HMGs) were identified in 1964 as non-histone proteins abundant in the chromatin structure. High mobility group box proteins are ubiquitous proteins shared by all higher animals and plants, and their primary structures are remarkably highly conserved among species. HMGBs are known to be abundant not only in nucleus but also in cytoplasm. The biological function of HMGs is still poorly understood. However, based on the finding that HMGs unwind the DNA double helix structure upon binding to DNA, it is thought that HMGs function as a versatile transcription-enhancing factor or nucleosome-unwinding factor in transcription by optimizing DNA conformation to enhance transcriptional activity.
Several types of HMGBs have been identified, including, for example, high mobility group box protein 1 (HMGB-1 or HMG-1), high mobility group box protein 2 (HMGB-2 or HMG-2), high mobility group protein 3 (HMG-3), high mobility group protein 8 (HMG-8), high mobility group protein 17 (HMG-17), high mobility group protein I (HMG-I), high mobility group protein Y (HMG-Y), high mobility group protein I(Y) (HMG-I(Y)), and high mobility group protein I-C (HMG I-C).
Furthermore, the present inventors analyzed the amino acid homology using genetic information analysis software “GENETYX” (SOFTWARE DEVELOPMENT), and found that human HMGB-1 exhibits 98.6% and 99.1% homology to bovine and porcine HMGB-1, respectively. Furthermore, human HMGB-1 shows 81.2%, 72.3%, and 79.4% homology to human, bovine, and porcine HMGB-2, respectively.
In 1999, Wang et al. for the first time quantified serum (blood) HMGB-1 by Western blotting using the polyclonal antibody which was prepared using HMGB-1 itself as an immunogen, and demonstrated that HMGB-1 could be used as a sepsis marker. In addition, they showed that it is possible to predict the death and survival of sepsis patients through precise measurement of blood HMGB-1. Specifically, Wang et al. describe that the survival rate was significantly improved in the sepsis model mice administered with an antibody against HMGB-1 as compared to the untreated sepsis model mice. This suggests that HMGB-1 not only serves as a potential sepsis marker but may also be involved in sepsis as a causative agent. In other words, HMGB-1 may be a mediator of sepsis. Since no definitive therapy is available for sepsis to date, the discovery described above is very important (Non-patent Document 1).
Furthermore, various reported data demonstrate that HMGB-1 is also induced upon inflammation and is thought to trigger the secretion of substantial amounts of various cytokines (Non-patent Documents 2 to 4), supporting that HMGB-1 can be a target in therapy. This suggests the significant potential benefit of precisely quantifying blood HMGB-1 and inhibiting the function of HMGB-1, instead of simply detecting blood HMGB-1.
Furthermore, the present inventors have found that HMGB-2 is often detected in biological samples where HMGB-1 is detected (Non-patent Document 5). It has also been revealed that HMGB-2 does not have HMGB-1's mediator activity in diseases, even though HMGB-2 shares high homology with HMGB-1 (81.2%) (Non-patent Document 6). Therefore, it is very important to specifically measure or inhibit HMGB-1 without any HMGB-2 influence.
Antibodies are very useful tools in measuring blood HMGB-1 concentration and inhibiting the activity of HMGB-1 as a therapeutic target. However, it would be difficult to obtain antibodies exhibiting high affinity to HMGB-1, which specifically bind to HMGB-1 but not to HMGB-2.
The reason is not just the extremely high homology between HMGB-1 and HMGB-2. In general, when preparing an antibody against an antigen of interest, animals that are easy to care for (pigs, rabbits, goats, sheep, mice, rats, and the like) are immunized with the antigen of interest. Generally, all kinds of efforts are put into immunization, such as the use of adjuvants, to induce high-affinity antibodies. However, this may induce an inflammatory response, which in turn induces HMGB-1 in the animal body. Such treatments cause extremely high stress on the animals being immunized.
The very high inter-species homology of HMGB-1 also makes it difficult to obtain anti-human HMGB-1 antibodies. Specifically, the primary structures of pig, bovine, goat, sheep, mouse, and rat HMGB-1 differ from that of human HMGB-1 in only two or three residues at the amino acid level (Non-patent Document 7). Accordingly, when such an animal is immunized with human HMGB-1, high-affinity antibodies induced in the animal are absorbed by HMGB-1 induced in the immunized animal, and as a result, the antibodies obtained will have reduced quality and low affinity.
Moreover, individual difference among immunized animals is also an important factor when obtaining high-quality antibodies. The present inventors actually experienced that only one of five rabbits gave useful antibodies against HMGB-1 because of individual differences. This phenomenon is highly reproducible.
Documents of related prior arts for the present invention are described below.    Non-patent Document 1: H. Wang et al., Science, (1999) 285: 248-251    Non-patent Document 2: Andersson, U et al., J. Exp. Med, (2000) 192: 565-570    Non-patent Document 3: Scaffidi et al., Nature, (2002) 418: 191-195    Non-patent Document 4: Park et al., The Journal of Biological Chemistry, (2004) 279: 27    Non-patent Document 5: Shingo. Y., Clini Chem, (2003) 9: 1535-37    Non-patent Document 6: Ueno. H., Am J Respir Crit Car Med, 2004    Non-patent Document 7: L. Wen, Nucleic Acids Research, (1989) 17: 1197-1214    Non-patent Document 8: H. J. Lachmann and P. N. Hawkins, Curr Opin Pharmacol., (2006) 6: 214-20